• informational
  Messenger RNA (mRNA) is the intermediate "courier" of the protein-coding genes of the DNA. It is a relatively short-lived template that binds to the ribosome and translated by the ribosome (and enzymes) into protein.

• functional
  Functional RNA is transcribed from specific genes, but never translated into protein. These are "finished products" that perform vital functions related to gene expression. These are encoded by a relatively small family of genes (a few dozen to a few hundred, depending on species).
  • Transfer RNA (tRNA)
    These three-dimensional molecules bind to amino acids and bring them to the ribosome for addition to the growing polypeptide chain.

  • Ribosomal RNA (rRNA)
    These are the major component of ribosomes, the protein/RNA complexes responsible for assembling proteins from the mRNA code. rRNA is relatively stable and long-lived. It accounts for the majority of the RNA present in the cell at any given time.

• Small nuclear RNAs (snRNA)
  These are involved in the processing of the mRNA transcript, and form components of the spliceosome that guides removal of introns and splicing of exons.

  Perhaps the most interesting RNA molecules are those that act to suppress gene expression, thus providing a mechanism for control of gene expression. A relatively large proportion of the eukaryotic genome is devoted to the genes encoding these functional RNAs.

  • Micro RNAs (miRNA) regulate the amount of protein manufactured by a cell at any given time, depending on internal feedback mechanisms in the cell.

  • Small interfering RNAs (siRNAs) and piwi-acting RNAs (piRNAs) both facilitate RNA interference.

    In nature, it is likely that RNA interference inhibits the reproduction of viruses, thus protecting the host cell.

    siRNAs (plants) and piRNAs (animals) both prevent the transposition of potentially damaging transposable genetic elements into new chromosomal loci in the host cell, protecting the host genome from disruption.

    Here is a comparison of siRNA and miRNA.

    And here's the world's cutest RNAi video.

Transcription and translation are happening constantly in any cell. Hence, rRNA, tRNA, mRNA and snRNA are constantly being transcribed (i.e., their transcription is constitutive).

The other functional RNAs (miRNA, siRNA, piRNA, lncRNA) manufactured only when they are needed. See a complete list of the types of RNA discovered so far.

Messenger RNA: Encoding Polypeptides The only type of RNA that carries the code for building proteins is mRNA. In the initial phase of gene expression, DNA is transcribed into mRNA, which is read by the ribosomes and translated into protein.
Proteomes The full range of proteins a particular organism is able to manufacture is known as the complete proteome.
A cellular proteome is all the proteins found in a specific type of cell (and/or tissue) under a specific set of environmental conditions.

Gene expression changes not only on a daily, hour-by hour basis, but also over the lifespan of an organism.
Even your daily Circadian Rhythm is ultimately under genetic control, as the hormones and other molecules that direct your body to perform daily functions are at least partly encoded by one or more genes that respond to daily cycles.

Transcribing mRNA in Prokaryotes No matter which type of RNA is being manufactured, the machinery of transcription is essentially the same.
Transcription is the process by which the DNA code is rewritten into the informational code of RNA.

The three phases of this process are known as initiation, elongation, and termination. Initiation When a gene is being transcribed, the nitrogenous base code must be made available for reading by polymerizing enzymes. But where to begin? Much of what we first learned about transcription came from our old pal, E. coli.

At the start of every gene, there is a short base sequence (about 60 base pairs (bp) long) known as the promoter. This is where the polymerizing enzyme will attach.

When conserved sequences are found in several different locations in the genome, or in homologous regions of genomes of different species, they are known as consensus sequences. These may have resulted from functional or evolutionary (i.e., common ancestry) relationships among the sequences.

Retention of conserved sequences suggests that these sequences have not tolerated a great deal o mutation: their products are sensitive to changes, and may not function in mutant form.

In E. coli and other prokaryotes, one such consensus sequence is found in the promoter of all genes: The Pribnow Box.

• The Pribnow Box, located approximately 10 base pairs upstream of first DNA base actually transcribed, facilitates proper attachment of RNA polymerase.

• The sequence is sometimes called the Pribnow-Schaller Box, named after its discoverers, David Pribnow and Heinz Schaller.

• The sequence is usually T(82%) A (89%) T(52%) A(59%) A(49%) T(89%), with the likelihood of the base shown in parentheses.
• The Box (a "box" is a short sequence of nucleotides with a specific function) notably consists of A-T pairs (two H-bonds), not G-C pairs (three H-bonds).

• When RNA polymerase is attached, less energy is needed to denature the double helix, opening it for transcription.

RNA polymerase must be able to

• Recognize start (initiation) and stop (termination) signals on the DNA.
• Avoid transcribing the non-informational DNA.

• core factor (made up of four protein subunits, α, α' β, and β')
  this region of the enzyme polymerizes the chain of RNA
• sigma factor
  The sigma (& sigma;) factor is a small protein that recognizes the promoter and facilitates binding of RNA polymerase to it. Bacterial species manufacture several different sigma factors (E. coli has seven). The specific sigma factor used to initiate transcription of a given gene depends on the gene and on the cell's internal environment.
  
  An RNA polymerase with its sigma factor removed is "blind" and attaches randomly to the DNA.

• holoenzyme - a biochemically active compound formed by the combination of an enzyme with a coenzyme.
• coenzyme - a small, not-proteinaceous, molecule required for activation of an enzyme

The DNA template strand to which the mRNA nucleotides are attached is known as the non-coding strand.

• The non-coding strand base sequence is complementary to the mRNA base sequence.
• antisense strand is also known as the antisense or template strand.

• The coding strand base sequence is the same as the mRNA base sequence.
• The coding strand is also known as the sense strand or non-template strand.

Elongation

Termination

• direct/intrinsic (a.k.a. "rho-independent")
• rho-dependent

Intrinsic/Direct Termination

• The mRNA transcript itself has a termination sequence about 40bp long.
• Within this section are inverted repeats--sections that read the same, forward or backward.

• When these are transcribed, they form complementary sequences that bind together, forming a stem loop:
  

• The "stem" is rich in the more stable G-C bonds.

• The DNA encoding the stem loop is immediately followed by a long string of Adenines. This is transcribed as a long string of Uracils.

• This less stable area A/U of the RNA/DNA hybrid causes the RNA polymerase to pause there.

• After the RNA polymerase transcribes the two complementary regions, the inverted repeats release from the DNA template and bind into a stem loop.

• Formation of the stem loop may reduce RNApol's affinity for the complex, facilitating its release.

Rho-dependent Termination

• In rho-dependent genes, a small hexamer (six-subunit) protein named rho recognizes the termination sequences on a nascent RNA.

• Rho-dependent genes do not form stem loops.

• Instead, they have a 40-60bp region rich in cytosines, poor in guanines, and containing a segment known as rut (rho utilization site).

• Rho protein binds to rut, which is just upstream from an unstable sequence on the gene that causes RNA polymerase to pause.

• The proximity of the rho protein allows it to actually knock the RNA polymerase off the strand, terminating transcription.

The Finished mRNA

• In prokaryotes, the primary mRNA transcript is translated, without modification, directly into protein, and the two processes occur simultaneously.
  

• In eukaryotes, the primary transcript is modified via processing and splicingmbefore traveling to the ribosomes for protein translation.
  • 5' end is capped with 5-methyl guanosine
  • 3' end is capped with a line of adenines
  • introns are removed, and remaining exons are spliced together

But this is only one of the many ways that eukaryotic transcription is more derived than prokaryotic transcription. Let us explore.

Eukaryotic Transcription: What's Different? A typical prokaryote has about 1000 genes, and one type of RNA polymerase to transcribe them all.

Eukaryotes have many more genes than prokaryotes, and also have a great deal of non-informational DNA interspersed between the genes. This means that the factors searching for promoters have a lot more searching to do before they connect. To facilitate this, eukaryotes have not one, but three different types of RNA polymerase:

• RNA polymerase I - transcribes nucleolar organizer RNA
• RNA polymerase II - transcribes protein-encoding genes (mRNA) and some snRNAs
• RNA polymerase III - transcribes 5S rRNA and tRNA genes

Quick Guide to Eukaryotic Transcription Acronyms

• CTD - carboxyl terminal (or "tail") domain - part of a general transcription factor (protein) in which the terminal end is a free carboxyl group (-COOH). The RNA polymerase II's CTD usually contains multiple (50+) repeats of the sequence tyr-ser-pro-thr-ser-pro-ser. The CTD serves as a binding site for other proteins that activate RNA polymerase II.
  The CTD:
  • participates in initiation transcription
  • caps the RNA transcript
  • attaches to the spliceosome (more on this later) to facilitate exon splicing

• GTF - general transcription factor - proteins that bind around the promoter, attracting RNA polymerase. Names are such as...
  • TFIIA
  • TFIIB
  • etc.
• TATA box - conserved sequence in the eukaryotic (and archaean) promoter region

• TBP - TATA-binding protein - a component of TFIID that binds to the TATA box

• PIC - pre-initiation complex - RNA polymerase bound to the appropriate GTFs

...(or very similar), usually followed by a short sequence of Adenines.

The Initiation Complex

• provides energy to drive the reaction
• changes its physical configuration, allowing it to move

Elongation in Eukaryotes
Elongation is essentially similar to that seen in prokaryotes, with the new RNA being laid down inside the transcription bubble (bordered by two Y-junctions) formed by the denaturation of the DNA.

Two major differences:

I:

• In prokaryotes, translation may begin at the 5' end of the new RNA strand while transcription is still taking place (translation is co-transcriptional).
• In eukaryotes, transcription and translation are both spatially and temporally separated.

II:

• Processing of the mRNA transcript is directed by the CTD, which does not exist in prokaryotes.

• The repeats serve as binding sites for enzymes that cap, cleave and splice the RNA.

• Recall that the CTD is located right where the new RNA squirts out of the enzyme, so it's conveniently placed to perform these functions.

• The CTD is alternately phosphorylated and dephosphorylated, and its changing composition affects its affinity for various processing proteins. It can sequentially determine which protein task must be completed as the mRNA transcript emerges!

• As the first bit of mRNA emerges from the RNA polymerase, guanyltransferase, guided by the CTD, attaches a 7-methylguanosine triphosphate cap to the 5' end. This protects the transcript from enzyme degradation, and is essential for translation.
• The A-U-rich conserved sequence (the polyadenylation signal) close to the 3' end is recognized as it emerges, and the CTD directs the associated proteins to cut the RNA about 20 bases down from the sequence.
• Finally, 150-200 adenines are attached to the cut end, forming a poly-A tail. This, too, protects the transcript from degradation.

Introns and Exons

• The nematode Caenorhabditis elegans has only about 12,000 - 13,000 known genes.
• Homo sapiens is believed to have about 25,000 functional genes. Only about twice as many.
• So how do we explain the ability of the human body to manufacture more than 100,000 different proteins from its protein-coding genome?
• The answer: exon shuffling.
• One gene can encode several different proteins if it is processed in alternative splicing such that the finished sequence is different when the cell requires a different protein product.

• Interesting side note: Not all species are capable of alternative splicing. Whereas alternative splicing appears to be rare in plants (they often have more genes than animals), it's now known that more than 70% of human genes can be spliced in different ways.
• Mutations that cause splicing defects (splice site mutations) can have mild to devastating consequences for the organism:
  • Type 1 neurofibromatosis
  • certain types of microcephaly
  • neuromuscular disorders
  • keratin/skin disorders
  • the list will grow as new relationships are discovered.

Intron Excision, Exon Splicing
How are the introns excised and the exons splice together?

Several models for intron excision/exon splicing have been proposed.

Self-splicing Introns

• Discovered in the ciliate Tetrahymena, group I introns are removed via this mechanism.
• A ribozyme is an RNA molecule that exhibits enzymatic activity.
• A hammerhead ribozyme (a.k.a. hammerhead RNA), is an RNA that can cleave itself via a small, conserved structural motif called a hammerhead (because of its shape).
  

• Introns are enzymatically snipped out by the ribozyme RNA itself.
• Hydrolysis of guanidine phosphate (GTP) molecules provides energy to drive the reactions.

• Ribozymes thus catalyze removal of their own introns via transesterifications reactions--breaking the phosphodiester backbone in an unusual way to create
  (a) a 2'-3' phosphodiester bond (causing the backbone to form a ring at its broken end)
  (b) a hydroxyl group at the 5' end (highly unstable and reactive) reacts to create a terminus that is stable and unreactive:
  
  • This configuration develops at both ends of the intron, preventing it from re-attaching to the exon.
  • Ribozymes can both hydrolyze themselves and form peptide bonds. They have been found in numerous different systems across organisms.
  • Could these properties be a window to our ancient past? The RNA world theory holds that because RNA is the only macromolecule know to both encode genetic information and act as an enzyme, it was likely the first genetic material found in living organisms. DNA came later.

• introns fold into secondary structure to form stem loops
• RNA autocatalyzes the breakage points, snipping out the introns
• These may be evolutionary remnants of snRNA's (small nuclear RNA's) involved in the splicing of nuclear precursor mRNA in the spliceosome

U1, U2, U4, U5 and U6

1. U1 binds at the 5' end of the intron to be removed.

2. U2 binds at a central location of the intron, known as the branch point.

3. Meanwhile, U4, U5 and U6 bind together to form a complex.

4. U4/5/6 complex approaches the intron; as it draws near, U4 releases from the complex; the U6 portion of the complex is now unstable and reactive.

4A. U5/6 complex replaces U1 at the 5' end of the intron.

5. U6 has a high affinity for U2; it binds to U2, creating a "loop" out of the intron. The two ends of the adjacent exons are now close together, though still separated by the intron.

6. U5 catalyzes the linkage of the two exons ends which have been brought into close proximity by the binding of U6 and U2.

7. U5 and RNA may both participate in intron removal; U5 does the actual catalysis of the final phosphodiester bond between the exons. So cool.

The spliceosome is one type of control mechanism in intron/exon splicing, allowing greater accuracy of splicing.

Conserved sequences in the genes being spliced are critical to the operation of the spliceosome. Three conserved sequences (one at each end of the intron, and one in the middle) are highly conserved across species, probably for functional reasons.

Since spliceosomes may physically vary, different introns may be removed from the same transcript, depending on the "needs" of the cell at the given time.

Result: a huge variety of products potentially coded by the same gene. (alternative splicing; exon shuffling)

Evolutionary Origin of Introns and Exons

• Archaebacteria (Domain Archaea), unlike true bacteria (Domain Bacteria), have introns and exons. Could this be evidence of common ancestry with eukaryotes? Or is it just convergence?

  Most recent evidence suggests that eukaryotes share a most recent common ancestor with the thermophilic archaeans, such as those found in hotsprings and deep sea hydrothermal vents.

  (Never heard of hydrothermal vents? Click on the pic!)

• Exon shuffling may have given early archaeans and--later--eukaryotes a competitive edge, allowing them to produce a great variety of proteins in a rapidly changing environment.

Oddities of Eukaryotic RNA: Editing

• substitution editing
• chemical alteration of a base in situ
• mostly seen in mtRNA, cpRNA and some nucRNA

• mini- and maxi-circles of DNA are present in the kinetoplasts (modified mitochondria) of Trypanosoma.
• These evidently code for a fifth type of RNA, known as "guide RNA" (gRNA).
• gRNA seems to "guide" the RNA editing process.
• Poly Uracil editing may explain why Trypanosoma is so readily able to constantly change its surface proteins, thwarting the host's immune system.